Plasma Membrane Level Research Articles

A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Regulation of the cell surface expression of chloride transporters during epileptogenesis

The process is commonly known as epileptogenesis refers to the cascade of molecular and cellular changes that transform the brain to make it hyperexcitable and capable of generate recurrent spontaneous seizures. Unfortunately, our understanding of the molecular changes that affect the brain during epileptogenesis remains incomplete. Recent evidence suggests that dysfunction of cation-chloride transporters (CCCs) might be one of the factors that contribute to the deficits in inhibitory neurotransmission observed during epileptogenesis. This study analyzed the cell surface expression of CCCs during epileptogenesis and during chronic epilepsy to evaluate if a loss of CCCs from the plasma membrane might contribute to hyperexcitability. Alterations in the plasma membrane expression of CCCs were mostly detected during the early phase of the epileptogenic period, suggesting that dysfunction of CCCs might contribute to the alterations in the chloride gradient previously detected. Together, the findings presented here suggest that aberrant regulation of the plasma membrane levels of CCCs might contribute to the impartment of GABAergic neurotransmission and that CCCs dysfunction might be relevant for the initial appearance of spontaneous seizures.

Erythrocyte senescence and membrane transporters in young and old rats

Alterations at the level of plasma membrane are reported to play an important role in cellular senescence. The present study was undertaken to correlate cellular senescence, membrane transport processes and organismal aging. To achieve this objective activities of membrane linked Na+/K + ATPase (NKA), Na+/H+ exchanger (NHE) and correlation with membrane hydrxyperoxide level, sialic acid content and membrane protein oxidation was studied in density-gradient fractionated young and old erythrocytes from 4 and 24 month old Wistar rats. The results reveal that cellular aging within the tissue is associated with significant decrease in activities of NKA and NHE of senescent erythrocytes in comparison to younger cell population of same age group. The result shows that impaired ion homeostasis due to altered membrane transporters including functional and compositional changes may be one of the reasons responsible behind rat erythrocyte aging.

Unsaturated amino acids derived from isoleucine trigger early membrane effects on plant cells

Unsaturated amino acids (UnsAA) have been shown to affect the activity of various biological processes. However, their mode of action has been investigated poorly thus far. We show in this work that 2-amino-3-methyl-4-pentenoic acid (C2) and 2-amino-3-methyl-4-pentynoic acid (C3) structurally derived from isoleucine (Ile) exhibited a multisite action on plant cells. For one, C2 and C3 induced early modifications at the plasma membrane level, as shown by the hyperpolarization monitored by microelectrode implantation in the pulvinar cells of Mimosa pudica, indicating that these compounds are able to modify ionic fluxes. In particular, proton (H+) fluxes were modified, as shown by the pH rise monitored in the bathing medium of pulvinar tissues. A component of this effect may be linked to the inhibitory effect observed on the proton pumping and the vanadate-sensitive activity of the plasma membrane H+-ATPase monitored in plasma membrane vesicles (PMVs) purified from pulvinar tissues of M. pudica and leaf tissues of Beta vulgaris. This effect may explain, in part, the inhibitory effect of the compounds on the uptake capacity of sucrose and valine by B. vulgaris leaf tissues. In contrast, an unexpected action was observed in cell reactions, implicating ion fluxes and water movement. Indeed, the osmocontractile reactions of pulvini induced either by a mechanical shock in M. pudica or by dark and light signals in Cassia fasciculata were increased, indicating that, compared to Ile, these compounds may modify in a specific way the plasma membrane permeability to water and ions.

Quantum Disentanglement: Electrical Analysis of the Complex Roles of Ions in Filling Vesicles with Glutamate

By expressing vesicular glutamate transporters at high levels in plasma membrane and applying voltage clamp methods, Eriksen etal. (2016) have identified a Cl(-) channel in the transporter that is coactivated by protons and Cl(-).

375. TDP-43 Interacts with Cannabinoid Type 1 Receptor Modulating Its Signaling Properties

To identify new molecular chaperones of cannabinoid type-1 receptors (CB1R), we used a proteomic approach in HEK293T cells, transfected with two spicing variants of this receptor, CB1R and CB1AR. These two isoforms differ only in the composition of the N-terminus domain. Surprisingly, the only protein found to interact differently with CB1R and CB1AR was transactive response DNA-Binding Protein 43 (TDP-43), which is known to be involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The specific interactions between CB1R and TDP-43 were also confirmed by co- immunoprecipitation not only in transfected HEK293 cells, as well at the endogenous level in rat crude spinal cord. TDP-43 overexpression decreased CB1R plasma membrane levels by stimulating receptor internalization, but it did not change the total receptor levels. Further, TDP-43 enhanced CB1R ubiquitination to similar levels, as induced by the full CB1R agonist, CP 55,940. Also, overexpression of TDP-43 resulted in reduced effects of CB1R stimulation on cAMP, P-ERK1/2, and P-CREB responses. Present results revealed TDP-43 as an unexpected molecular chaperone of CB1R and these findings may have significance in the regulation of CB1R intracellular trafficking and possibly in the treatment of ALS and FTLD.

Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning.

The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.

Rat white adipocytes activate p85/p110 PI3K and induce PM GLUT4 in response to adrenoceptor agonists or aluminum fluoride.

Adipocyte responses to adrenergic and ß-adrenoceptor(-AR) (adrenoceptor) regulation are not sufficiently understood, and information helpful for elucidating the adrenoceptor-responsive machinery is insufficient. Here we show by using immunoprecipitated kinase analysis with a phosphatidylinositol 3-kinase (PI3K) p85 antibody that PI3K activation was induced by treatment with 10 or 100 µM norepinephrine (NE) for 15 min or with 10 mM aluminum fluoride (AF, a guanosine triphosphate (GTP)-binding (G) protein activator) for 20 min in white adipocytes (rat epididymal adipocytes) and that treatment with pertussis toxin (PTX, a G-protein inactivator) inhibited PI3K activation induced by the 20-min treatment with AF in the cells. In addition, western blot analysis revealed that glucose transporter 4 (GLUT4) level in the adipocyte plasma membrane (PM) fraction was increased by treatment with 10 µM NE, 100 µM dobutamine (DOB, a ß1-AR agonist), or 0.1 µM CL316243 (CL, a ß3-AR agonist) for 30 min or with 10 mM AF for 20 min. NE or AF treatment triggered 2-deoxyglucose (2-DG) uptake into adipocytes under the above conditions. Our results advance the understanding of responses to adrenoceptor regulation in white adipocytes and provide possible clues for clarifying the machinery involved in adrenergic and ß-AR responses in the cells.

Microchamber Array Technology and Microdosimetry for Nanosecond Pulsed Electric Fields Cell Exposure

Nanosecond Pulsed Electric Fields (nsPEF) with megavolt-per-meter intensities have been increasingly studied over the last decade for their effects on biological cells and their potential biotechnological and biomedical applications. nsPEF have been successfully used to trigger effects both at the plasma membrane level (nanoporation, apoptosis, externalization of phosphatidylserine) and the internal cell structures (intra-cytoplasmic calcium release, mitochondria membrane permeabilization) affecting their performance and viability. Recently, developments have been made toward clinical applications of nsPEF such as tumor eradication and anti-cancer treatments. We have previously used molecular dynamics simulations and a quasi-static approach using a custom implementation of 3D finite-difference analysis to investigate the physical mechanisms of electropore creation (Biophysical journal, 104(2), page 250A). The exposure of biological cells to such short duration, high intensity fields, requires the development of specific instrumentation technology and numerical implementations.In this work, we propose a delivery system based on a microchamber array of interdigitated electrodes to investigate nsPEF effects on cells. The microchamber array composed of 60, 40 μm-spaced electrodes allows delivering nsPEF with durations of a few nanoseconds and intensities up to a few tens of MV/m. We also propose to study the transmembrane potential of biological cells and intracellular components using numerical and experimental, specifically developed microdosimetry techniques. A realistic exposure model was implemented where the cell has been set laying flat over a glass support between two parallel electrodes delivering the nsPEF. Three-dimensional realistic irregularly shaped cells model were constructed using cross-sectional planes obtained by confocal fluorescence imaging of human cells. An experimental microdosimetry technique using microfluorimetry was developed for temperature and transmembrane potential measurements at the biological cell level.

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR.

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models.

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Acid sphingomyelinase is required for cell surface presentation of Met receptor tyrosine kinase in cancer cells.

Receptor tyrosine kinases (RTKs) are embedded in the lipid bilayer of the plasma membrane, but the specific roles of various lipids in cell signaling remain largely uncharacterized. We have previously found that acid sphingomyelinase (ASM; also known as SMPD1) regulates the conserved DAF-2 (the ortholog IGF-1R in mammals) RTK signaling pathway in Caenorhabditis elegans How ASM and its catalytic products, ceramides, control RTK signaling pathways remain unclear. Here, we report that ASM regulates the homeostasis of Met, an RTK that is frequently overexpressed in various cancers. Inactivation of ASM led to a rapid loss of Met from the plasma membrane, reduced Met phosphorylation and activation, and induced Met accumulation in the trans-Golgi network (TGN). However, trafficking of integrin β3 and vesicular stomatitis virus glycoprotein (VSVG) was largely unaffected. Knockdown of syntaxin 6 (STX6) also blocked the Golgi exit of Met. Depletion of either ASM or STX6 led to aberrant trafficking of Met to lysosomes, promoting its degradation. Our studies reveal that ASM and ceramides, together with STX6 and cholesterol, constitute a new regulatory mechanism for the exit of Met from the Golgi during its biosynthetic route, which is used to rapidly replenish and regulate the plasma membrane levels of Met in various cancer cells.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca2+, and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants

Rhizosphere acidification is essential for iron (Fe) uptake into plant roots. Plasma membrane (PM) H+-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H+-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 (CBR1) increases the levels of unsaturated fatty acids, which stimulate PM H+-ATPase activity and thus lead to rhizosphere acidification. CBR1-overexpressing (CBR1-OX) Arabidopsis thaliana plants had higher levels of unsaturated fatty acids (18:2 and 18:3), higher PM H+-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbr1 loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H+-ATPase activity but higher rhizosphere pH. Reduced PM H+-ATPase activity in cbr1 could be restored in vitro by addition of unsaturated fatty acids. Transcript levels of CBR1, fatty acids desaturase2 (FAD2), and fatty acids desaturase3 (FAD3) were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H+-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake.

AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells.

AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity. In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues. To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl(-) absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the overexpression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level.

Abstract B145: PK/PD evaluation of an anti-CEACAM5 antibody drug conjugate, in a colon patient-derived-xenografted mice model

Abstract CEACAM5 is an oncofetal protein that is expressed in a number of tumors of epithelial origin such as colorectal cancer (CRC), lung and gastric cancer. Differential expression of CEACAM5 protein in tumor tissues compared to normal tissues make this target attractive for targeting with an antibody-drug conjugate (ADC). The ADC that is characterized in this study contains a specific anti-CEACAM5 antibody and the maytansinoid derivative DM4, a potent antimitotic agent that inhibits microtubule assembly. DM4 is covalently bound to the humanized IgG1 (hIgG) antibody through a N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB) linker that is stable in plasma and cleavable inside cells. After binding and internalization into CEACAM5-positive cancer cells, this ADC is degraded, releasing cytotoxic DM4 metabolites. In the present study the Pharmacokinetic (PK), Pharmacodynamic (PD) and efficacy relationships of this ADC were investigated against subcutaneous colon Patient-Derived-Xenograft (PDX) in severe combined immunodeficient (SCID) mice. The modulation of several biomarkers after a single administration of this compound at 3 dose levels were quantified in the tumor by immunoassay (phosphorylated Histone H3 (pHH3), a marker of cells arrested in mitosis and free human CEACAM5 expression). The spatial localization in the tumor of the biomarkers (pHH3, free human CEACAM5, DM4 and hIgG) was analyzed by immunohistochemistry (IHC). A population pharmacokinetic analysis of the ADC was performed first, followed by population PK/PD analysis for pHH3 induction and tumor growth inhibition (TGI). This study has shown both by immunoassay and IHC that a single administration of this anti-CEACAM5 ADC induced in the xenografted tumor a dose dependent increase of pHH3 expression compared to control, with a maximal induction observed at 24-48 hours. The levels of DM4 and hIgG detected by IHC in the tumor were dose dependent and progressively decreased with time. DM4 and hIgG part of the ADC were essentially immunodetected at the membrane level of tumor cells and sub-localization was observed in vesicular intracellular compartments at later time point which confirms ADC tumor cell penetration. The PK/PD relationship analysis indicated that compound concentration producing 50% of the maximum pHH3 stimulation was 2.3 μg/mL and the threshold concentration for tumor eradication was 8 μg/mL in this colon PDX model. Both immunoassay and IHC assessment confirmed that single administration of the ADC had no impact on CEACAM5 expression at the tumor cell membrane. Overall these data have confirmed mechanism of action of this anti-CEACAM5 ADC and its attractiveness as a potent compound to induce dose dependent CEACAM5 expressing tumor cell death. This compound is now being explored in a phase 1 clinical trial in cancer patients. Citation Format: Christophe Henry, Céline Nicolazzi, Céline Amara, Anne-Marie Lefebvre, Christelle Larois, Florence Attenot, Claire Brillac, Marion Bouillon-Pichault, Nathalie Fagniez, Stéphanie Decary, Cécile Combeau, Pierre-François Berne, Souâd Naimi, Marielle Chiron, Véronique Blanc. PK/PD evaluation of an anti-CEACAM5 antibody drug conjugate, in a colon patient-derived-xenografted mice model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B145.

Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis

Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis hasbeen established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipidcomposition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells.

Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

IntroductionSystemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration.MethodsWe used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration.ResultsOur in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3+-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3+ cells; (iv) down-regulated expression of adhesion molecules on endothelial cells.ConclusionsTaken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

Periapical lesions decrease Akt serine phosphorylation and plasma membrane GLUT4 content in rat skeletal muscle.

Periapical lesion (PL) promotes insulin resistance; however, the mechanisms underlying this alteration are not fully understood. Therefore, in this study, we aimed to evaluate the Akt serine phosphorylation status and GLUT4 expression levels in the gastrocnemius muscle (GM) of rats with PL. Male Wistar rats (n = 42) were distributed equally into control (CN) and PL groups. The pulpal tissue of the PL group rats was exposed to the oral environment for 30days. Thereafter, glucose and insulin levels were assessed, followed by homeostasis model assessment of insulin resistance (HOMA-IR). The Akt serine phosphorylation and GLUT4 levels of microsomal (M) and plasma membrane (PM) fractions were evaluated by western blotting and analyzed statistically. Compared to CN group rats, PL group rats had lower insulin sensitivity (as observed by HOMA-IR), lower Akt serine phosphorylation status after insulin stimulus, and lower GLUT4 levels in the PM fraction. However, the M fraction in the PL group did not differ significantly from that of the CN group. PL decreases insulin sensitivity, Akt phosphorylation, and PM GLUT4 content. The present study indicates that preventing endodontic disease can thwart insulin resistance.

ACTIVITIES OF PROXIMAL TUBULE ENZYMES AND ALBUMIN CONCENTRATION IN URINE OF CHILDREN TREATED WITH METHOTREXATE

In order to study methotrexate nephrotoxicity, the activities of proximal tubule epithelial cell membrane enzymes: alanine aminopeptidase (AAP) and gamma-glutamyltransferase (GGT), as well as of lysosomal N-acetyl-beta-D-glucosaminidase (NAG) and urinary albumin concentrations were determined in 12-h-urine samples of 30 patients with lymphoblastomous leukemia. The patients were i.v. receiving 4 individual methotrexate doses of 2000 mg/m 2 every 15 days followed by leucovorin as a protector. Control and methotrexate-treated group, each consisting of 30 examinees, included 4–10 years old children of both sexes. Statistically significant increase of AAP and GGT activities, expressed as U/mmol creatinine was observed after the first two ( p < 0.05), as well as after the remaining two therapies ( p < 0.01) in relation to the control. Enzymatic activities of these two enzymes decreased to control value before the second and the third methotrexate application, but they increased again after the third application and remained elevated up to the end of experiments. Significant increase of NAG activity expressed as U/mmol creatinine ( p < 0.01), as well as urinary albumin levels (mg/mmol creatinine; p < 0.01) were registered after the third methotrexate therapy and this elevation of the same statistical significance of the differences remained stable till the end of the therapy. Based on these results it can be concluded that during the time period of two first applications nephrotoxic methotrexate action is reversible and at the level of proximal tubule epithelial cell membranes. During the last two applications impairment is irreversible and at the level of cell organelles and glomerular filtration.